African trypanosomes are unicellular eukaryotic flagellates which cause serious diseases in man and livestock (sleeping sickness, Nagana). They have an intricate life cycle involving transmission between a mammalian host and an insect vector, the tsetse fly. Transcription in T.brucei and other kinetoplastida is characterized by a number of biochemical peculiarities. Among these is the observation that all mRNAs in trypanosomatids are generated by the joining of two separate RNA precursor transcripts via the process of trans-splicing. As a result, the 5' end of all mRNA in Tbn[unreadable]cei consists of a common, capped 39-nt non-coding mini-exon. Importantly, trans-splicing allows the 5' cap to be added to each mRNA post-transcriptionally. Trans- splicing may thus have uncoupled the production of the capped mRNA by RNA polymerase (pol) ll. Comparing the alpha-amanitin sensitivity of trypanosome protein- coding gene transcription revealed that the Variant cell Surface Glycoprotein (VSO) gene expression site (ES) and Procyclic Acidic Repetitive Protein (PARP or procyclin) genes are transcribed by an RNA pol that is resistant to alpha amanitin, a characteristic of transcription by RNA poll. A large body of indirect evidence, now available, supports the notion that RNA pol l, which normally only transcribes rRNA genes, transcrihes the VSG and PARP genes. This proposal presents a thorough and conclusive analysis of the RNA polymerases involved in,the transcription of VSO and PARP genes. First, the 5' end structure of primary transcripts will be charactenzed. This study will reveal whether RNA pol II primary transcripts are capped and whether an RNA polymerase associated capping activity is present in trypanosomes. Second, the finding of a capped 5' end or triphosphate or di-phosphate at the 5' end of the primary transcripts can unambiguously settle whether RNA pol I or pol II controls the transcription of VSO and PARP genes. Third, trypanosome poll point mutations the result in temperature sensitive mutants and trypanosome poll knock-out cell lines will be established. These cell lines will be used to further address the nature of the VSO BS and the PARP gene transcription. The success of this research will resolve the question, raised a decade ago, whether transcription of the surface coat protein genes of T. brucei is mediated by RNA poll. More importantly, the concept of temperature sensitive mutants will introduce a new genetic approach useful for a variety of biochemical and genetic studies of Mrican trypanosomes.